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Overview of Immunology

The field of immunology encompasses many different areas of study and therefore has a
very broad range of primary samples. Here we have outline some significant samples that
researchers are examining on a daily bases. They range from human primary and clinical
samples to immobilized cell lines to cell samples from murine models.

Although there is a great variety of clinical and primary samples, most often the
concentration and viability is determined by a single method: hemocytometer counting
using trypan blue. This counting method may not be optimal for all samples. Samples
such as bone marrow, cord blood, tissue digests, bronchoalveolar lavage, and other
primary and clinical samples may be contaminated with residual red blood cells, tissue
and cellular debris. These contaminants can lead to inaccurate enumeration of immune cells.

Nexcelom's solution is to perform fluorescent based cell analysis using acridine orange (AO)
and propidium iodide. Since AO and PI are nucleic acid dyes, they will bind to and label only the DNA of live
and dead nucleated cells. This method allows for the identification, enumeration, as well
as provides cell viability of nucleated cells in any sample.

Strategy for the detection of nucleated cells in complex primary samples

Sample Detection

Quantifying Cell Samples in 4 Easy Steps Using Cellometer Vision

With the Cellometer Vision, just 20µl of sample is added to the Cellometer Counting Chamber. Imaging and analysis of the samples is completed in less than 60 seconds. Bright field and fluorescent cell images can be viewed to check cell morphology and verify cell counting. Total cell count, concentration, and mean diameter are automatically displayed.

1. Pipette 20µl

2. Insert Slide

3. Select Assay & Click Count

4. Results in 30 seconds!

Human Clinical Samples

Bone Marrow (BM)

Bone Marrow (BM) is a complex tissue that is the site of hematopoiesis and B-cell development.
Multipotent, non-hematopoietic stem cells, such as mesenchymal stem cells, may be isolated from human
bone marrow. Furthermore, bone marrow mononuclear cells (MNCs) can be isolated by diluting the bone
marrow sample and performing a density gradient separation. After centrifugation, the MNC layer is
collected and may be further processed to isolate various subpopulations (hematopoietic progenitor cells,
mesenchymal stem cells, CD33+ myeloid cells, and CD138+ plasma cells) using cell sorting.

Current Issue: Sample contamination with red blood cells may lead to inaccurate enumeration of immune cells.

Current Solution: Lyse red blood cells prior to cell count.

Nexcelom Solution: Perform fluorescent based analysis. Using AO/PI allows for the identification, enumeration
of mononuclear cells, as well as provides cell viability in the counted sample.

Bone marrow samples can be processed and used for many different experimental applications such as studying
hematopoietic cell differentiation pathways and acting as controls for BM malignancies studies.

Total Number Nucleated Cells in Bone Marrow can be Measured Using AO

Isolated Mononuclear Cells

Bone Marrow Stem Cells - CD34+

F1F2

BR

Ratio FL / BR

Viability

MNC-BM_1

3296

4575

0.72

98.20%

MNC-BM_2

1388

1605

0.86

98.60%

MNC-BM_3

2008

2935

0.68

98.20%

Three human bone marrow samples were stained with AOPI and imaged. Acquired bright-field (BR) images were counted separately
from the acquired AOPI (F1F2) images. As shown in the table (left), many more particles were counted in the BR than in F1+F2. Since the count of F1F2 to BR are not 1:1,
the ratio of FL /BR reflect those counts. Furthermore, the tabulated ratios in the three separate experiments are shown to be different
from experiment to experiment. The ratio variation is the direct results of imaging of non-nucleated particles in bright-field. Due to
the complex nature of the sample, we conclude that counting nucleated cells in a bone marrow samples should be performed using AOPI
staining.

Cord Blood (CB)

Cord blood (CB) is collected at the time of delivery in a 250 mL standard blood bag containing anti-coagulating agents. CB-Mononuclear Cells (MNC) can be isolated by diluting the cord blood sample and performing a density gradient separation. After centrifugation, the MNC layer is collected and can be further processed to isolate subpopulations (hematopoietic progenitor stem cells, CD34+ neutrophils, CD14+ monocyte, CD19+ B cells, CD3+, CD4+, CD8+ T cells, and CD56+ NK cells) using cell sorting.

Cord blood samples can be processed and used for many different experimental applications such as treatment of hematopoietic, genetic and SCID (severe combined immunodeficiency) disorders.

Total Number Nucleated Cells in Cord Blood can be Measured Using AO

Isolated Mononuclear Cells

Cord Blood Stem Cells - CD34+

F1F2

BR

Ratio FL / BR

Viability

MNC-CB_1

2488

4659

0.53

98.10%

MNC-CB_2

2613

7152

0.37

94.10%

MNC-CB_3

2313

5791

0.40

93.30%

MNC-CB_4

1939

3449

0.56

96.70%

MNC-CB_5

2243

6450

0.35

95.80%

Five human cord blood samples were stained with AOPI and imaged. Acquired bright-field (BR) images were counted separately from the acquired AOPI (F1F2) images. As shown in the table (left), many more particles were counted in the BR than in F1+F2. The ratios of FL to BR show that in almost every sample there was more than double the amount of particles counted in BR compared to AOPI. Variability in ratios stems from counting non-nucleated particles in bright-field. We conclude that counting nucleated cells in complex samples such as cord blood should be performed using AOPI.

Cord blood samples can be processed and used for many different experimental applications such as:
an early detection tool for different diseases, studying metabolism and immunity pathways, and discovery
of potential therapeutic antibodies.

HPC (Human Hematopoietic Cells)

CD34+ Stem Cells

Murine Samples

Murine primary cells. Murine animal models are widely utilized to study different human diseases. A great
variety of samples are regularly collected and processed. Obtaining a cell count and viability are two common
processes that are part of standard laboratory practices.

Current Issue: During standard harvests, the processing of numerous samples very time consuming. Furthermore,
due to a great variety of potential samples multiple methods may be needed to determine cell count and viability.

Current Solution: Hemocytometer counting.

Nexcelom Solution: Perform fluorescent based analysis. Using AO/PI allows for the identification,
enumeration, and provides cell viability of nucleated cells in the collected sample. This procedure may
be performed on many different collected samples.

A large immunological study was performed where 30 individual samples of mouse lymphocytes and splenocytes
were collected and processed on the same day. Each sample was stained with AO/PI and imaged using a Cellometer
Auto 2000. The total cell count, concentration and viability were acquired for all of the samples.

Lym7

Lmy6

Lym2

Lym 5

Total cell count

49

1419

10635

24245

Live cell concentration

1.07E+05

2.84E+06

3.11E+07

6.75E+07

Viability

63.20%

58.00%

84.40%

80.30%

Bright field cell image

Counted live /dead cell image

Above, is the representative set of 4 different mouse lymphocyte samples (Lym7, 6, 2, 5). A bright-field and a counted fluorescent image are shown for each sample. The samples ranged in concentration from 1.07x105 (with 49 cells counted) to 6.75x107 (with 24245 cells counted) and viability of samples ranged from 58.0% to 84.4%. Because images can be saved during data acquisition, they can be used for documentation and visual verification of counted cells.

Below, are the tables containing the data for each of the 30 collected and processed lymphocyte and slpenocyte samples. Reported below are the cell counts, concentrations, and viability for live cells. The same imaging and counting parameters were used for all samples. Total imaging time for all 60 samples was approximately 30 minutes compared to total counting time using a hemocytometer was approximately 2 hours.

Examples of Tumor Digests

Human Melanoma Digest Sample Stained with AOPI

Murine Tumor Digest Samples Stained with AO/PI

Bright field and AO/PI overlay images (far right) show the labeling of nucleated cells only.

Tumor Cells with Lymphocytes

Lymphocytes were added to a suspension of tumor cells and stained with AO/PI. The sample data was acquired and images
automatically analyzed. The cell diameter histogram shows that there are two distinct populations in our sample: one
(in red) is the smaller ~6.0 microns in diameter and the other (in green) are the tumor cells averaging about 13 micron in diameter.

Bronchoalveolar Lavage (BAL)

Bronchoalveolar lavage (BAL) is a medical procedure in which a bronchoscope is passed through the mouth or nose
into the lungs and fluid is squirted into a small part of the lung and then recollected for examination. BAL sample
may contain lung and bronchial epithelial cells, macrophage, lymphocytes, and other immune cells. BAL samples from
humans or mouse may be analyzed on the Cellometer.

Current Issue: BAL samples contain cellular debris making it difficult to identify and count cells of interest.

Current Solution: Upon isolation and purification of immune cells, flow cytometry or manual counting is used to
count the cells.

Nexcelom Solution: Perform fluorescent based analysis. Using AO/PI allows for the identification, enumeration, and
provides cell viability of nucleated cells in the collected sample.

Examples of Bronchoalveolar Lavage Samples

These murine BAL samples were collected from multiple individual experiments. Each sample was stained with AO
and imaged. Above is a collection of bright-field, AO fluorescent (FL), and BR-FL overlay images.

Conclusions & Cellometer Selection Guide

Clinical primary samples are routinely used for biology, immunology, and cancer research. The diversity and the quantity of the samples often poses a challenge of counting and calculating the concentrations and viability of samples in a time efficient and accurate manner. The family of Cellometer instruments provides researchers multiple tools to perform fast and consistent counts of nucleated cells. Fluorescent staining of blood-based and primary samples allows for accurate identification and enumeration of nucleated cells even in the presence of potential contaminants. The chart below provides a quick reference guide to which Cellometer may be right for your lab!

Give us a call +44 (0) 161 232 4593.
Experienced Nexcelom Applications Specialists are available 9:00am to 5:00pm GMT to assist with selection of a Cellometer system.